Global Regulation of Nucleotide Biosynthetic Genes by c-Myc

The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis

Characterization of c-myc as a transcriptional repressor

grantor: University of TorontoThe c-'myc' proto-oncogene encodes a nuclear phosphoprotein, whose expression has been tightly linked to cellular transformation ' in vivo' and 'in vitro'. The strong tumorigenic potential of Myc is reflected in its biological activities. Myc can promote cell cycle progression, inhibit growth arrest in the form of quiescence and senescence, and abrogate cellular differentiation in a diverse array of cell types. In essence, the net effect of Myc is to enhance cell growth by promoting cell proliferation and inhibiting growth arrest. Myc is believed to mediate these diverse biological activities by acting as a transcription factor, inducing or repressing the expression of specific subsets of genes. Analysis of the function of the Myc-induced genes that have been identified, reveals that some of these genes are required for progression through the cell cycle, particularly the transition from G1 to S phase. This observation suggests that Myc may enhance cell growth through the induction of genes which are essential to cell cycle progression. In comparison, less is known about the importance or mechanism of Myc repression activities, due to the small number of genes that have been reported to be repressed by Myc. Identification of the subset of genes which are repressed by Myc will help us to understand the mechanism of Myc-mediated gene repression, and how the repression of genes by Myc can promote cell proliferation. The crux of my research has been to identify and characterize novel gene targets for Myc repression, with the intention of revealing novel Myc-dependent pathways to drive cells out of growth arrest and promote cell proliferation. We have focused primarily on three genes: the 'cyclin D1' gene; the Platelet-derived growth factor ß receptor ('pdgf-ßr') gene; and the growth arrest gene, ' gadd45'; analyzing their expression in response to Myc-activation. In contrast to previous results obtained in selected immortalized cell lines, we clearly demonstrate that in primary mouse embryonic fibroblasts, cyclin D1 expression is not repressed by Myc. Indeed, loss of cyclin D1 expression is only evident in cells which exhibit Myc-activation and lack retinoblastoma tumour suppressor protein expression. Our data further suggest that the decrease in cyclin D1 expression is an indirect effect of cellular transformation rather than a direct effect of Myc. In addition, we have identified and characterized the repression of 'gadd'45 and the 'pdgf-ßr' genes by Myc. Analysis of these Myc-mediated repression mechanisms revealed that unlike Myc-mediated transactivation, Myc-mediated repression is comparatively more complicated and can be mediated by multiple pathways. The precise nature of these repression mechanisms is currently under study. The identification of these genes as targets for Myc repression has revealed that Myc repression pathways serve an important role in the ability of Myc to regulate the cellular response to changes in the extracellular environment, to drive cells out of growth arrest, and aid in tumour progression. Thus, it would appear that we are at the dawn of a new era of Myc research. Deciphering the significance of Myc repression will help us to understand how both Myc transactivation and Myc repression cooperate together to regulate normal cell proliferation, through the induction of cell cycle progression and the inhibition of growth arrest.Ph.D

Microarray and Biochemical Analysis of Lovastatin-Induced Apoptosis of Squamous Cell Carcinomas

We recently identified 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway, as a potential therapeutic target of the head and neck squamous cell carcinomas (HNSCC) and cervical carcinomas (CC). The products of this complex biochemical pathway, including de novo cholesterol, are vital for a variety of key cellular functions affecting membrane integrity, cell signaling, protein synthesis, and cell cycle progression. Lovastatin, a specific inhibitor of HMG-CoA reductase, induces a pronounced apoptotic response in a specific subset of tumor types, including HNSCC and CC. The mediators of this response are not well established. Identification of differentially expressed genes represents a feasible approach to delineate these mediators as lovastatin has the potential to modulate transcription indirectly by perturbing levels of sterols and other mevalonate metabolites. Expression analysis following treatment of the HNSCC cell lines SCC9 or SCC25 with 10 µM lovastatin for 1 day showed that less than 2% (9 cDNAs) of the 588 cDNAs on this microarray were affected in both cell lines. These included diazepam-binding inhibitor/acyl-CoA-binding protein, the activated transcription factor 4 and rhoA. Because the biosynthesis of mevalonate leads to its incorporation into more than a dozen classes of end products, their role in lovastatin-induced apoptosis was also evaluated. Addition of the metabolites of all the major branches of the mevalonate pathway indicated that only the nonsterol moiety, geranylgeranyl pyrophosphate (GGPP), significantly inhibited the apoptotic effects of lovastatin in HNSCC and CC cells. Because rhoA requires GGPP for its function, this links the microarray and biochemical data and identifies rhoA as a potential mediator of the anticancer properties of lovastatin. Our data suggest that the depletion of nonsterol mevalonate metabolites, particularly GGPP, can be potential mediators of lovastatin-induced apoptosis of HNSCC and CC cells